Characterization of the β -barrel assembly machinery in Fusobacterium nucleatum

The Centers for Disease Control and Prevention’s 2019 Antibiotic Resistance Threats Report highlights more than 2.8 million antibiotic infections each year, with at least 35,000 deaths per annum attributed to antibiotic resistance. The CDC’s 2022 COVID-19 Impact Report emphasizes a 15% increase in hospital-acquired resistant infections between 2019 and 2020, many which are caused by Gram-negative bacteria, bacteria characterized by two encapsulating membranes. The limited treatment options for Gram-negative bacterial infections underscore the critical need for new strategies to combat these pathogens. The β-barrel assembly machinery complex (BAM) is a protein complex located in the outer membrane (OM) of Gram-negative bacteria, facilitating the folding and insertion of β-barrel outer membrane proteins (OMPs) into the OM. Inhibiting the function of this complex is lethal for Gram-negative bacteria, making BAM a significant and promising drug target.

Fusobacterium nucleatum is a Gram-negative pathogen that functions in the oral microbiome, interacting with multiple levels of biofilm colonizers. F. nucleatum causes oral infections and is linked to colorectal cancer, impacting treatment response and disease recurrence. The pathogenicity of F. nucleatum in both biofilm formation and in cancer involves OMPs whose biogenesis relies on BAM; however, BAM has not been characterized in this organism. The goal of our study here is to better understand the composition, structure, and function of BAM and its potential as a drug target for F. nucleatum. We first used bioinformatics analysis and proteomics to investigate the putative composition of the BAM complex in F. nucleatum. While the core component BamA was identified, there was a notable absence of other typical accessory proteins in this organism's genome. Therefore, we postulate that unlike other bacteria such as E. coli and A. baumannii, the biogenesis of OMPs in F. nucleatum is mediated solely by BamA without the need of accessory components.

To investigate how BamA can accomplish OMP biogenesis itself, we employed biophysical techniques to analyze the structure of FnBamA. We resolved the cryo-EM structure of FnBamA in complex with several Fabs which showed novel structural features not previously observed in bacteria. In these structures, FnBamA was found to contain four N-terminal POTRA domains arranged in a J-shaped conformation, rather than elongated. The Fab was found to bind primarily along POTRA 3 which likely stabilizes the unique conformation of the POTRA domains. The C-terminal 16-stranded b-barrel domain was observed as an inverted dimer, with the dimer interface mediated by direct interaction of the b1 strands along the lateral seam of both barrel domains. Additionally, we determined the X-ray crystal structure of the barrel domain alone which was found as a monomer. Measurements of the barrel domain of FnBamA reveal it has a different shape and size than is found in other BamA structures such as in E. coli. Together, these structural differences provide clues to how FnBamA alone may accomplish OMP biogenesis when additional components are required in other bacteria. Our ongoing studies aim to further characterize the molecular structure and function of FnBamA in conjunction with promising antibiotics and other putative BAM components if discovered.